Kevin J. Catt, MD, PhD, Head, Section on Hormonal Regulation
Lazar Krsmanovic, PhD, Staff Scientist
Albert Baukal, Research Assistant
Hung-Dar Chen, PhD, Adjunct Investigator
Hao Feng, MD, PhD, Postdoctoral Fellow
Lian Hu, MD, PhD, Postdoctoral Fellow
Po Ki Leung, PhD, Postdoctoral Fellow
Zhong Lu, PhD, Postdoctoral Fellow
Xing Yin, MD, PhD, Postdoctoral Fellow
Samuel Quaynor, BA, Postbaccalaureate Fellow
Robert Gustofson, MD, Clinical Fellow ¹

Our research addresses the receptors and signaling mechanisms by which peptide hormones activate functional responses in their specific target cells. We characterize structure-function properties, signal transduction, and cellular processing of specific G protein-coupled receptors (GPCRs). Our program focuses on GPCRs for the hypothalamic peptide hormone gonadotropin-releasing hormone (GnRH) and the vasoactive octapeptide angiotensin II (Ang II) and on intracellular signaling pathways that mediate GPCRs' cellular actions. The GnRH decapeptide mediates neural control of the pituitary gland and gonadotropin secretion, exerts autocrine actions on GnRH neurons, and is essential for normal reproductive function. The angiotensin octapeptide plays important roles in aldosterone secretion, control of sodium balance, and regulation of blood pressure. The octapeptide has also been increasingly implicated in the etiology of cardiac, vascular, and renal disease as well as in type 2 diabetes mellitus. To elucidate their signaling pathways and the mechanisms by which they regulate metabolic, secretory, and growth responses, these receptors and their functions are studied in normal and immortalized hypothalamic neurons, pituitary gonadotropes, adrenal glomerulosa cells, hepatic cells, and transfected cell lines.

Regulation of pulsatile GnRH secretion

Krsmanovic, Hu, Wada,2 Catt

The hypothalamic control of pituitary function depends on episodic secretion of GnRH released from the median eminence into the descending portal vessels to the anterior pituitary gland. Our previous studies on hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells) revealed that GnRH and other agonists can exert both stimulatory and inhibitory actions on GnRH secretion because of their receptors' abilities to activate single or several G proteins in a time- and dose-dependent manner. Such activation leads to differential regulation of the PLC/InsP3/Ca2+ and adenylyl cyclase/cAMP signaling pathways and of GnRH neuronal firing, thereby regulating the frequency and amplitude of pulsatile GnRH release. These actions can result from agonist-regulated sequential coupling of activated GnRH receptor to Gq, Gs, and Gi proteins or from activation of several receptor subtypes (e.g., 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7) by a common agonist such as serotonin. The ensuing signaling responses, in conjunction with their modulation of the spontaneous electrical activity of the GnRH neuron, contribute to the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro.

The paradigm also applies to activation of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor (LHR) in cultured hypothalamic GnRH neurons and GT1-7 cells in which treatment with LH or hCG transiently stimulates and subsequently inhibits cAMP production and pulsatile GnRH release. Pertussis toxin (PTX) prevents marked and delayed impairment of cAMP signaling and episodic GnRH release in GT1-7 cells. These cells, and LH/hCG-induced release of membrane-bound Gs and Gi3 subunits, are indicative of differential G protein coupling to the LHR. AP firing in identified GnRH neurons also initially increased and then decreased during LH treatment while PTX prevented the inhibitory action of LH on AP firing. RT-PCR analysis of GT1-7 neurons revealed the expression of G protein-gated inwardly rectifying potassium (GIRK) channels in these cells. The LH-induced currents were inhibited by PTX and identified by electrophysiological studies as GIRK currents. These responses indicate that agonist-stimulation of endogenous LHR expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. Our findings demonstrate that regulation of GIRK channel function is an important factor in gonadotropin-induced inhibition of pulsatile GnRH release. Furthermore, this Gi-mediated mechanism could account for the suppression of pituitary function in pregnancy and during ectopic hCG production.

GnRH neuronal signaling pathways

Shah, Neithardt, ³ Catt

The GnRH decapeptide is a primary regulatory factor in the neuroendocrine control of reproduction. It is released in an episodic manner from hypothalamic GnRH neurons, which we previously found to express GnRH receptors. An analysis of the signaling pathways by which autocrine GnRH stimulation promotes proliferative signals and cell survival in hypothalamic GT1-7 cells revealed that both GnRH and epidermal growth factor (EGF) caused rapid phosphorylation of the cyclic AMP response element binding protein (CREB) and BAD, a member of the Bcl2 family. The selective EGF-R antagonist AG1478 attenuated phosphorylation of these proteins by GnRH and EGF. Inhibition of PKC and Src abolished the stimulatory effects of GnRH but not those of EGF, consistent with a critical role of these signaling molecules upstream of the EGF-R. Activation of PKC also elicited such effects of GnRH. Consistent with the prosurvival and mitogenic effects of PI 3-kinase/Akt (PI3K/Akt) downstream of the EGF-R, inhibition of PI3K diminished the activation of these proteins following stimulation with GnRH, EGF, and PMA. Overexpression of dominant negative Akt attenuated agonist-induced phosphorylation of BAD but not of ERK1/2 and CREB. Furthermore, overexpression of wild-type RSK-1 caused enhanced basal as well as agonist-induced phosphorylation of CREB and BAD, indicating a critical role of RSK-1 in activating cytosolic as well as nuclear proteins. These data have revealed novel signaling mechanisms of GnRH-induced phosphorylation of CREB and BAD in GT1-7 neurons through transactivation of the EGF-R.

Although adrenergic receptors (ARs) are involved in the regulation of GnRH release from native and immortalized hypothalamic (GT1-7) neurons, the AR-mediated signaling mechanisms and their functional significance in these cells have not been defined. Stimulation of GT1-7 cells with the alpha1-AR agonist phenylephrine (Phe) caused phosphorylation of MAP kinases (ERK1/2) mediated by PKC-dependent transactivation of the EGF-R. Phe stimulation caused shedding of the soluble ligand heparin-binding EGF (HB-EGF) as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP with CRM197 or of HB-EGF by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses to Phe but not to HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), the antioxidant N-acetylcysteine attenuated the Phe-induced phosphorylation of the EGF-R. These findings indicate that activation of the alpha1-AR causes phosphorylation of ERK1/2 through PKC, ROS, and Src and shedding of HB-EGF, which binds to and activates the EGF-R.

Cell-specific phosphatidylinositol 3-kinase signaling

Shah, Neithardt, Catt

Agonist-activation of many GPCRs causes phosphorylation of MAP kinases through transactivation of the EGF-R, leading to increased cell survival and growth, motility, and migration. Phosphatidylinositol 3-kinase (PI3K) is one of the major cell-survival signaling molecules activated by EGF-R stimulation. The extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation was determined by analysis of the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. GT1-7 neuronal cells express endogenous GnRH-R, the activation of which causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 lysophosphatidic acid (LPA) and of EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells did not depend on PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-Rs caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in the HEK 293 cells showed partial and complete dependence on the EGF-R and PI3K, respectively. The data demonstrate that the dependence of GPCR-induced ERK1/2 phosphorylation on PI3K varies according to cell type and that the involvement of PI3K during ERK1/2 activation is not necessarily associated with agonist-induced transactivation of the EGF-R.

Agonist-induced signaling in adrenal glomerulosa cells

Shah, Baukal, Catt

The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of GPCRs and receptor tyrosine kinases (RTKs). In many cell types, GPCR-induced MAPK activation is mediated through transactivation of RTKs, in particular the EGF-R. However, the extent to which such cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa cells express receptors for LPA and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/PKCs alpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), the EGF-R, protein kinase B/Akt, ERK1/2, and p90 ribosomal S6 kinase, the ERK1/2-dependent protein. Overexpression of dominant negative mutants of Ras or the EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, matrix metalloproteinase (MMP) and heparin-binding EGF did not impair phosphorylation. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in C9 hepatocytes was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in an MMP-dependent or -independent manner in individual target cells. Such phosphorylation reflects the ability of GPCRs expressed in cell lines and neoplastic cells to use distinct signaling pathways that can elicit responses that differ from those of native cells and tissues.

Constitutive activity of the angiotensin AT1 receptor

Nikiforovich, Catt

Molecular modeling suggested a possible molecular mechanism for the constitutive activity of mutants of the AT1R at position 111. A cascade of conformational changes in spatial positions of side chains along the transmembrane helix (TM3) from L112 to Y113 to F117 results in conformational changes in TM4 (residues I152 and M155), leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis as well as with correct predictions of the constitutive activity of mutants L112F and L112C. We also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable to that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined owing to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT1R reveals a novel, nonsimplistic perspective on the general problem of constitutive activity and clearly demonstrates the inherent complexity of the process of GPCR activation.

Angiotensin II signaling and pathogenic actions

Hunyady, Catt

Ang II-induced AT1R activation stimulates phospholipases A2, C, and D and activates InsP3/Ca2+ signaling, protein kinase C isoforms, and MAPK via Gq/11 as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (arrestins, GPCR kinase-interacting protein 1, p130Cas, paxillin, vinculin), RTKs, and the NF-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling systems, such as beta-arrestin-mediated MAPK activation and the Jak/STAT pathway. In addition to their roles in the physiological control of blood pressure, thirst, and sodium balance, these responses exert diverse pathological actions in cardiovascular, renal, and other cell types. It is noteworthy that locally generated Ang II, rather than the circulating octapeptide, often initiates such deleterious effects of AT1R activation. In heart failure, the harmful effects of aldosterone exceed those of the RAS. AT1R-mediated overproduction of reactive oxygen species exerts potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify AT1R's signaling in cardiovascular cells, leukocytes, and monocytes. Agonist-induced activation of AT1R also promotes the development of metabolic diseases and can increase tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is broadening the clinical applications of angiotensin-converting enzyme (ACE) inhibitors and AT1R antagonists in addition to their established therapeutic actions in essential hypertension. Furthermore, recent reports suggest that intracellular Ang II can exert actions on cell growth that are independent of the AT1 receptor.

In addition to the many examples of GPCR-mediated activation of the EGF-R and other RTKs, increasing evidence points to the converse effect of agonist-activated RTKs on specific GPCRs. We have observed that the agonist-activated EGF-R caused phosphorylation of the AT1R that was mediated by the EGFR, PKC, and PI 3-kinase and reduced both membrane-associated AT1Rs and inositol phosphate responses to Ang II. These effects were dependent on caveolin-1, which was endogenously phosphorylated and distributed in plasma membrane patches that underwent redistribution during Ang II stimulation. The process involved the agonist-induced phosphorylation and association of caveolin 1 with the AT1R, suggesting a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. Our studies demonstrated that agonist stimulation promotes the formation of a caveolin-dependent signalplex that includes GPCRs, RTKs, and probably other signaling proteins. The complex could facilitate protein phosphorylation, which mediates Ang II-induced transactivation of the EGF-R and activation of ERKs, and EGF-induced InsP3 accumulation and phosphorylation/desensitization of the AT1R. Our work has provided further insight into the nature and complexity of signaling via GPCRs and RTKs and the importance of caveolin and plasma membrane compartmentalization in signal transduction.

¹ Robert Gustofson, MD, Reproductive Biology and Medicine Branch, now in private practice in Colorado
² Keiko Wada, MD, PhD, former Postdoctoral Fellow, now at Asahikawa Medical College Hospital, Asahikawa, Japan
³ Adrienne Neithardt, MD, in private practice in Pennsylvania

COLLABORATORS

László Hunyady, MD, PhD, DSc, Semmelweis University of Medicine, Budapest, Hungary
Nadia Mores, MD, Catholic University, Rome, Italy
Gregory Nikiforovich, PhD, DSc, Washington University, St. Louis, MO
J. Alberto Olivares-Reyes, PhD, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico
Bukhtiar Shah, DVM, PhD, Division of Physiology and Pathology, Center for Scientific Review, NIH, Bethesda, MD

For further information, contact cattk@mail.nih.gov.